Apparatus for automated analysis

ABSTRACT

An apparatus for pretreatment of a sample of whole blood in a discrete fluid analyzing instrument comprises automated means for handling and analyzing the sample and means for performing a pretreatment step on the sample or a sub-sample of the sample. The means for pretreatment are used for immobilizing at least one substance or analyte from the sample or sub-sample wherein the substance or analyte is reversibly immobilized. Usually, the apparatus further comprises means for eluting the substance or analyte from the capture means prior to analysis.

This nonprovisional application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 61/515,992 filed on Aug. 8,2011 and 61/623,082 filed on Apr. 12, 2012 and under 35 U.S.C. § 119(a)of Patent Application Nos. 20115785 filed in Finland on Aug. 8, 2011 and20125401 filed in Finland on Apr. 12, 2012. The entire content of all ofthe above applications is hereby incorporated by reference. The presentinvention relates to method and apparatus for automated sample treatmentfor automated discrete fluid analyzer

BACKGROUND OF THE INVENTION

Several chemical analyses are done by automatic analyzing equipment inhospitals and laboratories. The level of automation varies, but presenttrend is to streamline the analyzing work as much as possible. As manymore complicated analyses must be performed manually by highly trainedlaboratory personnel, the productivity and throughput of such laboratorytesting services has been low. By increasing the automation capabilitiesof the laboratory, more of the work can be performed inside theautomatic systems without involvement of highly skilled personnel.Ideally laboratory assistants simply load samples in racks into theanalyzing apparatuses freeing laboratory chemists and biologists tofocus on interpreting the results and managing the operation of thelaboratory. Such a system provides good throughput combined with highcertainty and quality of analytical results.

Automated instruments for various kind of chemical analysis have beenwidely used for decades. They may combine both routine and sophisticatedassay techniques such as spectrophotometry, fluorometry, time resolvedfluorometry, chromatographic methods etc. with automated sampledispensing from original sample containers. Automation is often requiredas test workload increases within healthcare and clinical laboratories,public or commercial research and service institutes, or industrialprocess control.

Automatic discrete analysis techniques use computer controlledautomation to perform steps similar to those of manual methods. Forexample, a computer controlled robotic arm may be used to position aprobe of a pipette to aspirate or deposit a fluid sample, buffer orreagent into or out of any of a plurality of sample receptacles, aplurality of reagent receptacles and a plurality of reaction cuvettes.This is a “discrete” analysis technique because each sample is depositedin a discrete reaction cuvette, which is then subjected to an assayingdevice such as colorimetric photometer, or the like. Typically severaldiscrete analyses may be done on subsamples divided from a main sampleplaced on a receptacle. For example, from a serum sample, a panel ofserum lipids (triglycerides, cholesterol and HDL-cholesterol) can bemeasured.

Many sample types, particularly human samples for clinical testing, arenot ready for assay as collected and, instead, require a pretreatmentstep. For example, plasma or serum must be separated from whole blood;whole blood must be hemolyzed to release intracellular components;fecal, sputum or solid tissue samples must be homogenized and suspendedin liquids. In many cases of human excretions or other biological andindustrial materials, interfering proteins must be precipitated, oranalytes extracted from the original sample matrix. So while automaticanalyzers have significantly improved throughput rate of analyticaltesting service, manual sample pretreatment has become a primarybottleneck in daily work flow.

Manual processes not only add to the time and labor required to produceresults but also increase the risk of errors. Errors during theanalytical process have been addressed primarily by automation asautomation standardizes pipetting and dispensing steps, and eliminatesvariation in timing and differences between individual technicians.Sample pretreatment, however, remains prone to problems and wouldbenefit from automation as well.

To increase sample throughput and provide process standardization, someautomated or semi-automated sample pretreatment instruments have beendeveloped. These are mainly meant to be used in connection with highlysophisticated analysis techniques that require sample purification stepsbefore the actual measurement can be performed.

For example, TurboFlow™ columns (Thermo Fisher Scientific) are used toseparate small molecules from proteins or other large molecules in asample before introduction to a LC/MS system. For PCR techniques,desired cell types must often be selected from complex sample mixturesfor further treatment, and sample DNA or RNA must be purified tosubstantially eliminate background contamination before the actualcycling process. Semi-automated pretreatment instruments like thosebased on magnetic particles, e.g. the KingFisher™ instrument (ThermoFisher Scientific), are useful for such processes.

Publications WO 2004096443, US 2005069913, U.S. Pat. Nos. 5,985,153 and7,897,337 disclose examples of automated pretreatment methods andapparatuses and fully automated analyzing processes includingpretreatment of all input samples. These are mainly used for DNA and RNAanalysis wherein pretreatment is needed for all samples before analysiscan be performed. Contamination risk is also extremely high. Usuallydisposable sample containers are needed, as well as disposable pipettetips or dispensers for transporting samples, sample aliquots, analytesand reagents.

However, many laboratories must function without such sophisticatedautomated or semi-automated equipment. Typically, the main dailyworkload of many clinical laboratories is performed using low-costautomated analyzers based on simple analytical techniques such asphotometry or spectrophotometry. Such analyzers usually employconventional assay methods that provide result levels traditionallyconsidered sufficient for measurement of clinically significant ranges.Many compromises concerning acquired information and accuracy are madein order to achieve acceptable cost and speed while providing ease ofuse and maintenance.

Sample pretreatment is often considered as a separate process from theactual assay and, therefore, not well suited to instruments streamlinedto perform one type of analytical process or measurement quickly andefficiently. Pretreatments are designed according to sample matrix andnature of analyte, and are usually not bound to particular assayprinciples. This may explain the lack of pretreatment automation, assample oriented additional steps preceding the chemical assay reactionwould lower throughput and cause difficulty in timing of assaysequences.

Despite difficulties in combining pretreatment processes with automaticdiscrete analysis, some pretreatment systems have been combined toautomatic analysis apparatuses. Some of these systems are describedbelow for reference.

On-Board Pretreatment of Whole Blood in Discrete Photometric Analyzersin the Measurement of Glycated Hemoglobin A1c (HbA1c).

In on-board methods to measure HbA1c, a sample of whole blood ishemolyzed within a discrete photometric analyzer to release thehemoglobin molecules from within blood cells. Hemolyzation is carriedout by mixing whole blood with hemolyzing reagent in a reaction vesselor a cuvette and incubating the mixture for a defined time. Afterhemolyzation, an aliquot of the mixture is sampled to measure HbA1ccontent with a turbidimetric inhibition immunoassay and another aliquotis sampled to measure total hemoglobin content. In such method, theanalytes and matrix components are not separated prior to the analyzing.This kind of analysis can be performed by Konelab™ clinical chemistryanalyzers (Thermo Fisher Scientific), for example.

Cadmium Column Reduction of Nitrate

In a cadmium column, nitrate (NO3-) is reduced to nitrite (NO2-) usingCd2+ granules as catalyst. The reduction is accomplished by aspiratingsample to the column where the reduction takes place and then elutingthe reduced sample to a vessel. From the vessel an aliquot is sampled tomeasure nitrite. This kind of analysis can be performed by an Aquakem™analyzer (Thermo Fisher Scientific). In this system analytes undergo achemical reaction during the pretreatment; there is no means forseparating reacted from unreacted or other non-target analytes. Thenitrate reduction to nitrite can also be performed in a reaction vessel.

US 2009/0162942 discloses an automated discrete fluid sample analyzerthat includes a sample preparation module. The sample preparation moduleincludes a well configured to receive a sample deposited by a pipetteand a sample preparation device in fluid communication with the well.The fluid sample is transferred from the well to the sample preparationdevice which prepares the sample by using catalyst, ultraviolet light orheat. The target analyte is not separated from the sample.

Samples of whole blood may have to be pretreated also in order torelease a target analyte from components within the sample that bind orsequester the analyte. This can be done by preparation of a secondaryspecimen i.e. blood plasma or serum by centrifugation or filtration. Ahemolysis reagent like saponin or detergent may be added followed bycentrifugation or filtration. Likewise a denaturing agent may be used,e.g. acid (HCl, TCA) or an organic solvent (methanol, acetonitrile) or acombination of methanol and ZnSO, followed by centrifugation. Theseprotocols involve manual steps which are difficult to automate orintegrate into an on-line analysis procedure. In order to increase thedegree of automation, an automated on-line hemolysis processor referredto as a Bloodlyser® device has been described (Morello, R. et al. TherDrug Monit 29:143, 2007). As used, a sedimented blood sample is mixed byan autosampler procedure And the homogenized sample is pumped through astainless steel capillary heated to 75° C. and the resulting heat causesno precipitation of blood/plasma proteins yet results in completedisintegration of blood cells. This leads to generation of new matrixreferred to as “cell-disintegrated blood” by the developers of theprocess.

SUMMARY OF THE INVENTION

The present invention provides a novel approach to arrange integratedpretreatment for automated discrete analyzing apparatus.

According to one preferred embodiment of the invention a whole bloodsample is hemolyzed and at least one matrix substance or analyte isretained from the sample volume by capture means wherefrom it isreversibly removable.

According to embodiments of the invention, the hemolysis is performed byuse of: (i) capillary device at room temperature capable of applying ashearing force to physically rupture cellular components, (ii)sonication, (iii) detergents or other chemicals to disrupt cellmembranes, (iv) mild heat, followed by processing using an affinitycolumn or other preparatory column capable of capturing the analyte ormatrix component(s) or (v) using a heated capillary device.

According to one preferred embodiment, the matrix substance or analytethat is retained, is eluted or regenerated from the capture means formeasurement.

According to one preferred embodiment, analyte is retained.

According to one embodiment of the invention, the analyte is retained bya method that is chemically inactive in relation to the analyte that ismeasured from the pretreated volume.

According to another aspect and embodiment of the present invention, theinvention provides a method wherein the analyte to be measured isretained by immobilization.

According to one specific feature of the invention, the inventionutilizes a surface that immobilizes the substance that is to bemeasured.

At least one embodiment of the invention enables to enrich analytes.Enrichment is needed when the concentration of analytes in the sample istoo low for the measurement. This can be done by retaining the analytesin, for example a column, or other suitable apparatus, and eluting themin a volume that is smaller than the initial volume.

At least one embodiment of the invention enables the separation ofanalytes and matrix components that interfere with the analytemeasurement. This is done either by retaining the analytes in a columnor other suitable apparatus, and washing matrix components out beforeanalyte elution, or by retaining the matrix components in the column andwashing the analytes out before matrix elution.

According to one preferred embodiment of the invention, an integratedpretreatment step is provided comprising selectively either apretreatment wherein at least one matrix substance is retained from asample volume without chemically changing the chemical composition ofthe analyte or the sample is left untreated, whereafter one or moreanalysis are performed on the sample.

According to one embodiment of the invention, the measurements areperformed with a discrete fluid analyzer that performs chemicalreactions and photometric measurements in single/separate wells.

According to one embodiment of the invention, the measurement is atleast one of the group comprising photometric measurement orelectrochemical measurement.

According to one especially preferred embodiment, at least two differentmeasurements are performed on subsamples of one sample; either after apretreatment is performed, without performing the pretreatment or both.

According to one embodiment of the invention, dispensing andtransportation of subsamples of an analyte sample volume is performedwith a non-disposable or an on board washable dispenser or dispensertip.

According to one embodiment of the invention, at least one measurementis performed on pretreated or not pretreated subsample (sample aliquot)without reacting the subsample chemically.

According to one embodiment of the invention, at least one measurementis performed on pretreated or not pretreated subsample (sample aliquot)after adding at least one reagent thereto.

The above mentioned embodiments may be used at least for:

-   -   separating analytes from complexing or binding molecules,    -   separating analytes from cell debris,    -   separating analytes from substances interfering with analyte        detection,    -   desalting the sample,    -   exchanging the sample solvent,    -   separating analytes from matrix substances.

At least one embodiment of the invention is applicable to fractionationof analytes into different fractions. This is done by retaining theanalytes in the column or on other substrates, and eluting the analytesin fractions by using eluents with increasing elution strength.

Isolation and/or concentration of analytes from a sample can be achievedby using one of the following methods, or any combination of two ormore, including but not limited to:

-   -   immobilization/retention by use of:    -   1) antibodies or other specific binding substances,    -   2) passive adsorption,    -   3) covalently binding substances, or by filtering    -   concentration via immobilization and elution into reduced        volume,    -   immobilization of a non-target substance from the sample to        increase assay specificity,    -   immobilization of a substance from the sample to avoid        interference with assay performance,    -   partial purification of a sample by immobilization and elution        of a substance from a complex mixture of substances,    -   separation of a target analyte from cell debris,    -   separation of an analyte from complex forming or binding        molecules,    -   separation or purification of derivatives of the analyte.

The modes by which the retention is accomplished include, but is notlimited to, reversible covalent bonding, ionic interaction, sieving orfiltering (molecular size and shape, diffusion), polarity,hydrophobicity, molecule or group specific interaction and chiralrecognition. One aspect of the invention is to provide a novelhemolyzing process and apparatus wherein the whole blood sample ishemolysed by using a capillary device, for example, a plastic or glasscapillary tube, at the prevailing temperature of the sample, usuallywithin a range of 5° C. to 40° C. and more commonly at room temperature,capable of applying a sheering force to physically rupture cellularcomponents.

According to alternative embodiments of the invention, the requiredshear force can be accomplished by using a screening plate or successiveplates with small holes, a circular or straight slot between elongatedwalls or a ceramic, fiber or other type of a filter.

One aspect of the invention is to use capillary devices with an internaldiameter of about 1 mm, which are generally suitable for applying ashearing force sufficient to rupture cellular components with a bloodsample.

The invention, in one aspect, is an automated discrete fluid analyzingdevice providing sample pretreatment wherein at least one substance ofthe sample matrix or analyte is retained.

In another aspect the invention is an automated discrete photometricanalyzing device providing sample pretreatment wherein at least onesubstance of the sample matrix or analyte is retained.

According to one embodiment, at least one substance is retained byimmobilizing it to a solid support and eluting it from the solid supportfor analysis and/or measurement.

According to one embodiment the retained substance is removed from thesample and not subjected to analysis.

According to one embodiment of the invention at least one substance isretained from the sample by a filtration method, such as membranefiltration or a gel filtration.

According to one embodiment of the invention, the at least one substanceis retained by immobilization on a solid support.

According to one embodiment of the invention, the at least one substanceis retained by immobilization by an antibody.

According to one or more embodiments of the invention, the at least onesubstance is retained by one or more of following methods: reversiblecovalent bonding, ionic interaction, sieving or filtering (molecularsize and shape, diffusion), polarity, hydrophobicity, molecule or groupspecific interaction and chiral recognition.

Examples of Sample pretreatment process Sample contains Analyte(s) ofinterest (A), Interfering (I) and Other substances (O) and water orother Fluid (F). In this invention, upon user request by software,subsample is taken and automatically pretreated to bring the unalteredanalyte of interest (A) to the discrete photometric analysis.Component(s) Component(s) retained in removed from the pre- the pre-treatment treatment Samples for the discrete photometric No Sample unitunit analysis 1 A + F + A I + F + Analyte is concentrated and/orpurified in I + O (Analyte) O the pretreatment phase. In the measurementphase analyzer can automatically calculate its original concentration inthe sample. Analyte remains intact throughout the process. 2 A + F + IA + F + Sample is pretreated to clean the interfering I + O (InterferingO substances. Interfering substance(s) (I) are substance) retained inthe pretreatment unit. Unaltered analyte is transferred e.g. into asecondary sample holder and further directed to analysis. Thereafter thecapturing device is regenerated and elution and regeneration liquids arediscarded. Analyte of interest (A) can be calculated to the originalconcentration in the sample after the discrete photometer measurementfor the specific analyte. 3 A + F + Sample is Sample can also beanalyzed as such without I + O not directed the pretreatment step to thepre- treatment unit 4 A + F + Two or more pretreatments in any order I +O

Other objects and features of the invention will become apparent fromthe following detailed description considered in conjunction with theaccompanying drawings. It is to be understood, however, that thedrawings are intended solely for purposes of illustration and not as adefinition of the limits of the invention, for which reference should bemade to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows diagrammatically an automated discrete fluid analyzingdevice to which the invention is implemented.

FIG. 2 shows diagrammatically one embodiment of a pretreatment unit fordevice in FIG. 1.

FIG. 3 shows a flow diagram of one operation workflow of the invention.

FIG. 4 shows an explanatory screen display of an apparatus utilizing theinvention.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

In the subsequent text the following terms and devices should beunderstood to be defined as below:

Pretreatment

Separate step from analysis chemistry; provides e.g. a concentrated,fractionated or purified analyte. In general pretreatment may bemodifying or non-modifying in relation to the substance to bepretreated.

Discrete Fluid Analyzer

Discrete fluid analyzer performs chemical reactions and photometricmeasurements in single/separate wells. Usually an analyzer containsmultiple wells in which different reactions are ongoing simultaneously.Several different tests may be requested from each sample. Alsodifferent types of samples may be analyzed simultaneously.

Column

Column is typically a cylindrical or other formed vessel to which liquidsamples and reagents may be introduced and analytes and/or othercomponents eluted. The column may be in-line connected to a pump orsyringe in which case the samples and reagents are pumped to and fromthe column using positive or negative pressure. Or the column may befreely diffusing in which case samples and reagents are drawn to andfrom the column by gravity or by centrifugal force. The column may bepacked or lined with material that has affinity towards the analytes ormatrix components. As an alternative, the column may be packed withmaterial that functions as a sieve or filter. Physical treatments mayalso be connected to the column.

The column may be single use (disposable) or reusable.

Sample

A whole sample as collected is a “sample” or “primary sample”. The partof the sample that is pretreated, is a “sub sample”.

The substances that are analyzed are analytes. All other components in asample are matrix.

The analytes may include, for example: ions, molecules, groups ofchemically related substances, substance, family of substances such asproteins and chemical species.

Matrix includes all matter that is not measured, for example: celldebris, substances interfering with analyte measurement, analytecomplexing/binding substances that interfere with analyte measurement.

Room temperature is considered to be standard laboratory roomtemperature (RT) of 25° C.

Washable

Washable means herein a device that can be successively used by cleaningit between consecutive operative steps, opposite to a single use devicethat is discarded after every contact with analyte or other liquid inorder to avoid contamination.

FIG. 1 shows a typical discrete analyzer. As these apparatuses have beenin laboratory use and their operation principles are known to personsskilled in the art or can be easily studied from operating manuals andbasic literature, only a cursory description of the apparatus shown inFIG. 1 is provided herein as an example. The invention is not limited toany specific type of an automated discrete analyzing device as long asits operation is controlled by sample handling rules permitting samplehandling routines for each sample so that a request to analyze a samplemay include an assay of the measurements required and a request forpretreatment if such is desired.

FIG. 1 shows the main elements of an analyzing device. These arearranged on a suitable frame and include diluent water container 1 andwaste water container 2. Samples to be analyzed are placed on samplewheel 4 and reagents, enzymes and other media needed for analyses onreagent wheel 3. Samples are handled with sample dispenser 5 andreagents with reagent dispenser 6. Measurements of analytes orcomponents within the samples occur in incubator 7 with a photometer 8.

For the pretreatment of a sample, a pretreatment unit 9 is arranged inrelation to sample wheel 4. The sample wheel 4 is divided in sectionsthat form racks 10, 11, 12 arranged as reagent rack 10 for elution andwash liquids, microplate rack 11 for pretreated samples and sample racks12 for untreated samples. Between the sample and reagent wheels is acuvette dispenser 13. FIG. 1 represents an exemplary arrangement withother arrangements possible.

In further description of FIG. 1, when pretreatment is not needed forthe indicated measurement, cuvettes are fed from the cuvette dispenser13 to the incubator 7 whereafter sample volumes of the collected sampleare aspirated with the sample dispenser 5 into the cuvettes as requestedby analyzing assay request of the sample. Accordingly, reagents neededfor requested measurements are dispensed by reagent dispenser 6 into thecuvettes and after a measurement specific incubation period, themeasurement is performed for each cuvette by photometer.

When the analyzing assay request includes pretreatment, a portion of thesample is aspirated in the pretreatment unit 9. A side view of unit 9 isshown in FIG. 2.

The exemplary pretreatment unit of this embodiment is built on a frame14 that is rotatably mounted on the analyzing device. The main parts ofthe pretreatment unit may include a pump for sample aspiration andejection through line 17. Line 17 connects the pump line to apretreatment column 16. In order to enable use of a long column 16 andsufficient pretreatment time and to keep the dimensions of thepretreatment unit 9 small, the column shown in this embodiment is on agrooved stationary wheel 18. On the opposite end of the pump 17, thecolumn is connected to a suction or pipetting tube 15 for aspiration andejection of the sample.

One type of pretreatment column 16 is a column containing internalsurfaces that immobilize the analyte from the sample to the surfaces ofthe column. These surfaces may be walls of the column itself or somefiller material, which may be in particle or fibrous form. Forpretreatment in such a column the substance must be first retained andthen released from the surfaces and placed into cuvettes in formeasurements. For this the sample wheel has a reagent rack 10 forelution and wash liquids. Further, FIG. 1 shows a washing station 19arranged in vicinity of pretreatment unit 9. When a pretreatment isneeded, the cycle may start with a washing cycle of the column 16 at thewashing station 19, but usually the column 16 is washed at the end ofthe pretreatment cycle. Washing is performed simply by aspirating wateror other washing liquid into the column so that it is substantiallyfilled and then ejecting the washing solution into a waste container 2.The aspiration and ejection is repeated as many time as needed to obtaindesired level of purity and the washing liquid may be pumpedreciprocatingly in order to increase removing effect.

For pretreatment a desired amount of sample liquid is aspirated into thecolumn 16 through pipetting line 15 and the sample is pumped through thecolumn so that at least the entire sample volume has entered the column.This forms a sub-sample of the original sample, which remains untreatedand may be used for other measurements without the effects ofpretreatment. In the column, the substance, for example a molecule, isretained and immobilized on the wall or other interior surface of thecolumn. For this, the sample may be kept in the column or pumpedreciprocatingly in the column to ensure sufficient reaction time.Heating or cooling of the sub-sample may be used for enhanced effect.After the reaction time needed to immobilize the target analyte haspassed, the sample is ejected from the column. The target analyteremains immobilized within the column. In order to make the measurement,the immobilized molecule or substance must be eluted from the surfacevia washing the column with an elution liquid placed in the reagent rack10. The elution liquid is ejected together with the substance elutedfrom the column 16 to a microplate rack 11 to await transfer to theincubator. The measurement of the sample occurs in a routine manner, forexample, by a photometer according to operating principles of theanalyzing apparatus. Thus, the pretreatment step is merely oneadditional handling step in the analyzer for the sample and can bearranged within the normal measuring cycle without disturbing theoperation of the apparatus or changing the function, construction oroperation of the apparatus. The pretreatment unit and washing stations,reagents and elution liquids may be fitted to existing apparatuseswithout difficulty.

The main problem with handling samples for discrete analyzers is thesmall volume of the sample. Since the volume of the sample is small,many traditional pretreatment methods like evaporation or treatmentsbased on chemical reactions performed on the substance to be measuredare difficult to implement. The present invention provides a solution tothis problem by using capture or immobilization methods for eitherenriching the substance that is measured or its proportion in thesample. This is accomplished by retaining the target analyte orretaining other substances that interfere with the measurement of thetarget analyte and removing them from the sample. The retaining methoditself may vary, but the immobilization method discussed above providesone embodiment suitable for implementation for many variousmeasurements.

Another approach to retaining a substance is adsorption or physicalbonding of the substance to a surface or another substance via chemicalbinding. In practice binding may occur through covalent bonds,electrostatic interactions or van der Waals forces or other surfaceforces between molecules or substances.

Various pretreatment methods will be discussed below in further detail.

This method of using a pretreatment unit enables transfer of the treatedsample into the measurement unit. Transfer is controlled by a userfriendly software whereby the instrument user may simply add the primarysample tube in the instrument sample feeding unit and program thedesired assay. When a sample pretreatment step is necessary, thesoftware automatically directs the sample into the pretreatment unit,while other analyses from the same sample or other samples continue tobe performed without delay or disturbance.

Within the clinical and non-clinical fields, the present inventionbenefits numerous assays.

For example, many physiologically important compounds including withoutlimitation, Vitamin A, D and E, are fat-soluble and/or bound incirculation to a corresponding binding protein or other proteinstructures. Serum vitamin D samples must be pretreated in order todisrupt the protein complex and extract the analyte in an organicsolvent such as acetonitrile or methanol by a manual procedure. With apretreatment unit containing a surface for immobilizing the complexformed, the analyte can be eluted by a suitable solvent and thentransferred to the assay performing unit. Immobilization may beaccomplished by antibody binding or by chemical binding and the surfacewill be automatically regenerated and washed between samples. A similarseparation step may be adapted to many small molecular compounds andthus avoid using instrumentation more complicated than a photometer.

The pretreatment surface may comprise a coated surface inside a glass ormetallic column, enabling solid phase extraction. Particles of uniformor different size may be coated and packed in a tube or other columncontainer. Coated fibers or other materials may also be used. Thereactive surface may also comprise a microfluistic platform or otherwisecomprise liquid pathways molded or printed on various suitablematerials. The coating may include antibodies or other specific binders.Also, many chemically reactive surfaces (hydrophilic, hydrophobic,lipophilic, nitrogenous or organic group binding) may be useful. In oneembodiment, binding is irreversible or accomplished via bonds from whichthe substance can be removed and the surface regenerated or comprisesequilibrium binding.

Sophisticated and time consuming chromatographic techniques are oftenused to measure components in mixtures, if there are no simple ways toseparate the required analyte from the other constituents. In thesecases, the pretreatment unit may comprise a surface coated with anantibody against the analyte which is measured by a standard photometricmethod.

On the other hand, pretreatment may be used to purify the sample byimmobilizing a cross-reacting substance using an antibody against thesubstance that is coated on the pretreatment surface. The purifiedsample can then be measured by standard turbidimetry or other methods.

Within some immunoassays antibodies may cross-react with substancesrelated to the desired analyte. Pretreatment to remove suchcross-reactants may avoid such problems and antibodies or other bindingor marker substances with lower specificity may be used. Such anapproach may lead to reductions of the costs of antibody development.

An important application of the invention are assays that today cannotbe performed by photometric methods because of their low sensitivity.For instance, the preferred hormone to diagnose thyroid disorders,thyrotrophin-stimulating hormone (TSH), is typically so low in serumconcentration that complicated techniques usually performed by separateinstruments are used for measurements of TSH. If, however, animmunoaffinity pretreatment method for TSH is used to concentrate TSHapproximately 10-fold or more, a useful turbidimetric assay is possible.

For food safety, analysis of various toxic mycotoxins in agriculturalproducts is very important. For concentration and separation from samplematrices, extractions and immunoaffinity steps are necessary prior toassay. These steps may be automated by using specific antibodies ormycotoxin group binders for solid phase extraction in the pretreatmentunit.

A similar approach is useful in environmental analysis when measuringcyanotoxins, produced by Cyanobacteria.

In the brewery industry, certain iso-alpha-acids are monitored duringthe brewing process to estimate bitterness of beer. The reference methodused employs extraction with iso-octane. This procedure may be omittedand the assay automated by using pretreatment where the targetiso-alpha-acids are bound to a surface and then eluted by a suitablesolvent, to be transferred to measurement unit.

Many drugs of abuse (DoA) are tested in urine samples. This testing isfrequently hampered by analyte related molecules or drug metabolitesthat cross-react with the employed antibodies, causing false positiveresults. With a suitable pretreatment the interfering molecules can beremoved and a reliable assay performed thereafter.

On the other hand, many DoA assay suffer from lack of sensitivity whichmay lead to false negative results. Here, the urine sample can bedirected into a column where drug molecules are retained, and theneluted with a smaller volume than the original samples. Thus bettersensitivity and higher reliability is achieved.

One aspect of the invention is to enrich, purify or fractionate one ormore analytes to be measured from a sample by retaining methods likeimmobilization, sieving or filtering. Immobilization may be done onsurfaces or by antibodies so that desired substances or matrixcomponents are retained. Filtering, for example by membranes, may beused to separate and retain molecules of desired size. Cell separationcan be performed by flow-through microchannels. Pretreatment may bedivided or arranged in two separate phases including pretreatments suchas: reagent addition+incubation to form complex, reagentaddition+incubation with proteolytic or other enzymes to be removedlater, removal of lipids (lipemia) and removal of hemoglobin or otherinterfering substances. In one embodiment, the substance or substancesthat are to be measured are not chemically or physically reacted,modified or altered so, that the information content of a small sampleused in discrete analysis is maintained as unchanged as possible.Alternatively the invention is based on picking desired substances fromthe sample in order to enrich the sample itself or, preferably theanalyte to be measured directly. Different substance may requiredifferent pretreatment means, but the apparatus may be designed so thatthe pretreatment means such as columns can be easily changed accordingto assaying needs. The columns or other means may be disposable orreusable, and changed automatically.

One example of two-phase pretreatment involves a first step ofprocessing of a sample to release a target analyte from componentswithin a sample that bind or sequester the analyte, thereby increasingaccessibility to the target analyte, followed by a second step ofseparating the target analyte from matrix as described in herein. Anembodiment of such a two-phase pretreatment process is hemolysis of awhole blood sample, for example by use of: (i) capillary device, forexample a plastic or glass capillary tube with an inner diameter ofabout 1 mm, at room temperature capable of applying a sheering force tophysically rupture cellular components, (ii) sonication, (iii)detergents or other chemicals to disrupt cell membranes, or (iv) mildheat, followed by processing using an affinity column or otherpreparatory column capable of capturing the analyte or matrixcomponent(s).

In instrument and operational point of view some typical ranges andstructures can be contemplated. However, the invention is not limited tobelow presented features. regarding operating temperatures: 37 degreeswould be typical, and the range can theoretically be as wide aspresented above. In practice, it would be extremely difficult to havethe temperature equal to sample or room temperature as the temperatureinside analyzer always differs from outside. The hemolyzing capillarycould preferably be thermostated and standardized to a certaintemperature. The capillary itself would probably be a stainless steel,polymeric or glass capillary. The inner diameter can be 1 mm, but alsoas small as 0.1 mm or less. The shape of the capillary could be a veryshort capillary or tube-like device with one or more tiny orifices, oneafter the other, where the sample is pressed through.

Using a shear force caused by a capillary is advantageously performed atroom temperature or a temperature that is lower than disintegrationtemperature of the cells in the sample. For example compared to a heatshock method this method avoids any danger of disintegrating or damagingsubstances that are supposed to remain intact in the sample that isfurther processed or analyzed. Thus, hemolysis by shear force providesbenefits over other methods described herein.

Instead of conventional capillary, a capillary slot formed between twoparallel planar surfaces or a stack of such surfaces could be used. Thiskind of a structure provides a straight capillary slot. If the slot orslots are formed between concentric tubes, circular slots are formed.Further, it can be contemplated that the cross section of the slot orslots needed are formed to have any desired shape, but a simple slot orcapillary would be probably easiest and cheapest to produce. Onepossible alternative is to use straining plate or successive plates withsmall holes and pump the sample through such structure. Even a fibrous,ceramic or other type of filter giving desired flow resistance thatcauses the required shear force might be feasible and easy tomanufacture and replace as necessary.

Mechanical blood damage (hemolysis) has been considered to be adisadvantage in various contexts. It is known that blood cellsdisintegrate under mechanical stress. An experimental study between celldisintegration under laminar and turbulent flow has been conducted andthe results are reported on ASAIO Journal 2004, Effects of TurbulentStresses upon Mechanical Hemolysis: Experimental and ComputationalAnalysis. The idea of the invention is to use mechanical hemolysis inadvantage as a pretreatment of blood samples.

The hemolysis effect of the capillary on treated blood cells in thesample depends on several factors. Some of these are the velocity of theflow, degree of turbulation, length of the capillary, diameter of thecapillary and the material of the capillary. Since these variable eacheffect of others mentioned, definite values of dimensioning thecapillary is difficult to give and dimensions given herein are onlyexplanatory.

In a discrete analysis several measurements may be done from a singlesample. To accomplish this using the present invention, a sub-sample isremoved from the primary sample and the pretreatment is done on thesubsample, leaving the primary sample unchanged. Thus, the primarysample may be used for further analyses of other substances.

One aspect of the invention is to combine above described pretreatmentmethods with a discrete analysis apparatus. The idea is thatpretreatment step operates integrated with the analyzer so thatpretreatment can be performed only when the analyzer receives such atest request that includes a pretreatment step. The pretreatment stepoperates independently from the analyzer so that normal analyzingprocesses can be done simultaneously with the pretreatment. The analyzerperforms requested measurements for each sample or subsample (samplealiquot) regardless whether the sample/subsample has been pretreated ornot. Also, some of the subsamples of one sample analyte may bepretreated and some not, exactly as required. As the pretreatment stepdoes not interfere with the measurements performed by the analyzer,several measurement cycles may occur simultaneously in the analyzer.

According to one valuable aspect of the invention, the inventionutilizes a discrete fluid analyzing apparatus, wherein measurements aredone photoelecmetrically or electrochemically on samples placed inseparate wells. Reactants are used as needed to induce changes in theanalyte in order to obtain response that can be measuredphotoelecmetrically or electrochemically. This type of measurementsystem is relatively inexpensive. Therefore inexpensive ways to handlethe analyte samples and subsamples are preferably used. For example,washable dispensers are used instead of disposable ones. This limits theinvention to measurements wherein contamination, cross-contamination ormixing of samples can be avoided to a level that does not interfere withthe measurements by washing and cleaning the dispensers or other fluidtransfer means, such as dispensing needles, probes or washable pipettetips.

The sample or a subsample taken from it undergoes changes duringmeasurement step when reactants are used. Therefore the molecule(analyte) to be measured must not change chemically under thepretreatment step. This is a requirement for the pretreatment step tooperate properly with a discrete analyzer.

FIG. 3 shows an example of some workflows that can be accomplished bythe invention. First, a sample 1 is obtained. The sample may be anyliquid sample, for example beer, wort, blood or urine. The sample isidentified by a data carrier such as a RFID chip or bar code and whenthe sample vessel enters the analyzer, data is read, or the sample canbe identified to the system manually by the user, the sample isidentified and the operating system of the analyzer requests allpretreatments and measurements that are to be performed on the sample.The sample may be placed on queue or taken directly forwards. Now thewhole sample or more commonly one or more subsamples are taken from thesample by a metallic needle and the extracted liquid is placed onmeasurement wells, such a s cuvettes.

If pretreatment is needed, the sample or subsample/aliquot istransferred to pretreatment step and pretreatment is performed asearlier has been described. In short, pretreatment includes capturingand releasing molecule that is to be measured in order to purify orenrich it. See description of pretreatment process for further details.Pretreatment should not change the chemical composition of the analyte.The pretreated sample is transferred to a measurement step. Someexamples are shown in FIG. 3. In Application 1, the sample (herein awhole sample, alternatively a subsample) can be measured photometricallywithout any chemical reaction. In this example, pretreatment is part ofthe Application and is automatically generated by requesting theApplication 1. In practice this application consists of pretreatmentstep with flexibility to design pretreatment parameters and the actualmeasurement step. In this case measurement is performed with no reagentadditions. Application 1 can be for example beer/wort bitterness orpolyphenol analysis.

Applications 2 and 3 include dispensing at least sample aliquot(subsample), dispensing reagent, incubation and photometric analysis. InApplication 3, two or more reagents are used (reagents 2, 3, 4, and 5).

When pretreatment step is not needed, sample or sample aliquot can beprocessed according to Applications 4-7. Now the sample/subsample(aliquot) is measured photometrically without reacting with a reagent(Application 4), measured electrochemically without adding reagent(Application 5) or by similar methods as in Applications 2 and 3(Applications 6 and 7). The Applications shown here are examples onlyand any measurement process application available in analyzer may beused in conjunction with both pretreatment steps, i.e. together withactual pretreatment or directing the sample or subsample directly tomeasurement step.

Applications 2-7 can be, for example, Beta-Glucan, Glucose, pH,conductivity, hemoglobin, HbA1C analysis, Ammonia or sulfatemeasurements.

Several samples, and their replicates, can be assayed simultaneouslywith several applications.

Sample dispensing does not require disposable tips. Vice versa,disposable tips should be avoided in order to keep the operating costsat bay. Application examples can have flexibility for volumes,dispensing orders and include e.g. additional sample blankings.

FIG. 4 shows one operating principle of the invention using anexplanatory display template. Herein the process starts when clientrequests a test. This request can be delivered to the analyzer by amemory device or made manually when a sample is loaded to the analyzer.When a test is requested for a named sample, the analyzer automaticallyselects a pretreatment step including pretreatment or no pretreatmentaccording to the specifications of the test as well as measurements,reagents, incubation time, mixing and other features. All parameters maybe determined flexibly.

The left side of the display shows a list of test and treatmentsavailable. Herein a test Bitter 300 is requested (highlighted). The listshows that the test is photometric and pretreatment is on use. On theright side is information about the test which shows that pretreatmentmethod is Bitter AU. Further details about reagents, samples, incubationand such used in a particular test are placed under tabs with samenominations (now shown in this display). The flow of the test isdisplayed on the right side of the tabs.

As can be seen on the display, Bitter 300 request performs automaticallya Bitter AU pretreatment (selection: Pretreatment procedure: Bitter AU)and thereafter a photometric test typical to a discrete analyzer. Theparameters of the photometric test are placed under the tabs bearingnominations of the parameters.

Multiple tests may be performed on one sample. As can be seen on theleft side of the display, the analyzer is capable of performing multipledifferent tests (see test name) simultaneously and some of the tests mayinclude pretreatment and some not. A pretreatment step can also beperformed without a following photometric test requiring additionalreagents. Usually, a subsample of the original or pretreated sample isneeded for each test.

Various embodiments of the method according to the invention are capableof performing optional, on demand pretreatment, simultaneous measurementand pretreatment, and/or measurement. Pretreatment may be performed on awhole sample or subsample or sample aliquot of the whole sample. Severaltests may be performed on a single sample. Sample or sample aliquot canalso be automatically treated or e.g. adjusted for pH by reagentaddition before pretreatment.

Numerous embodiments of the present invention have been shown anddescribed with attention paid to fundamental novel features of theinvention as applied to the embodiments. However, it is understood thatvarious omissions and substitutions and changes in the form and detailsof the invention may be made by those skilled in the art withoutdeparting from the spirit of the invention. For example, it is expresslyintended that all combinations of those elements which performsubstantially the same results are within the scope of the invention.Substitutions of the elements from one described embodiment to anotherare also fully intended and contemplated. It is also to be understoodthat the drawings are not necessarily drawn to scale but they are merelyconceptual in nature. It is the intention, therefore, to be limited onlyas indicated by the scope of the claims appended hereto.

The invention claimed is:
 1. An apparatus for automated pretreatment ofa sample of whole blood in a discrete fluid analyzing instrument, saidapparatus comprising: a hemolyzer capable of hemolyzing at least asubsample of the whole blood, a pretreatment unit capable of retainingat least one substance or analyte from the subsample by immobilizing theat least one substance or analyte on a surface of the pretreatment unit,and a dispenser capable of eluting the at least one substance or analyteretained by the pretreatment unit, wherein the hemolyzer is selectedfrom at least one of the following: (i) a capillary device operable at atemperature below disintegration temperature of cells in the sample andcapable of applying a sheering force to physically rupture cellularcomponents, (ii) sonicator, (iii) a detergent applicator capable ofapplying detergents or other chemicals to disrupt cell membranes, or(iv) a heater capable of applying mild heat to the whole blood, and theapparatus is capable of operating the hemolyzer, pretreatment unit anddispenser such that hemolyzation, pretreatment and elution are performedin an automated fashion.
 2. The apparatus according to claim 1 whereinthe pretreatment unit comprises capture means capable of immobilizingthe at least one substance or analyte.
 3. The apparatus according toclaim 2 wherein the pretreatment unit is capable of reversiblyimmobilizing the at least one substance or analyte.
 4. The apparatusaccording to claim 2 wherein the capture means is chemically inactive inrelation to the at least one substance or analyte.
 5. The apparatusaccording to claim 2 wherein the capture means is regenerable.
 6. Theapparatus according to claim 1 wherein the pretreatment unit is capableof reversibly immobilizing the at least one substance or analyte.
 7. Theapparatus according to claim 1 further comprising a measurement unitcapable of measuring at least one property of the at least one substanceor analyte retained by the pretreatment unit.
 8. The apparatus accordingto claim 7 wherein the apparatus is capable of simultaneously orconsecutively directing non-pretreated samples or non-pretreatedsubsamples to the measurement unit.
 9. The apparatus according to claim7 wherein the measurement unit is capable of performing at least one ofelectrical measurements and photometric measurements.
 10. The apparatusaccording to claim 7 wherein the measuring unit is capable of performingat least two different measurements on the sample simultaneously orconsecutively.
 11. The apparatus according to claim 7 wherein themeasuring unit is capable of measuring at least one property of thehemolysed subsample in a first measuring step, and simultaneously withor consecutively to the first measuring step, directing thenon-pretreated sample portion to a second measuring step to measure atleast one property of the non-hemolysed sample portion.
 12. Theapparatus according to claim 1 wherein the hemolyzer is a capillarydevice capable of hemolyzing at a temperature below disintegrationtemperature of blood cells.
 13. The apparatus according to claim 12wherein the apparatus is capable of ensuring that hemolyzation occursprior to immobilizing the at least one substance or analyte.
 14. Theapparatus according to claim 1 further comprising an affinity column orpreparatory column capable of capturing the at least one substance oranalyte.
 15. The apparatus according to claim 1 further comprising atleast one washable dispensing means for transferring and handling the atleast one substance or analyte.
 16. The apparatus according to claim 1wherein the hemolysed subsample comprises a first volume, thepretreatment unit is capable of retaining the analyte and the dispenseris capable of eluting the analyte at a second volume that is smallerthan the first volume.
 17. The apparatus according to claim 1 whereinthe at least one substance is an analysis interfering substance and thedispenser is capable of eluting and discarding the analysis interferingsubstance.
 18. An apparatus for automated pretreatment of a sample ofwhole blood in a discrete fluid analyzing instrument, said apparatuscomprising: a hemolyzer capable of hemolyzing at least a subsample ofthe whole blood, a pretreatment unit capable of retaining at least onesubstance or analyte from the subsample by immobilizing the at least onesubstance or analyte on a surface of the pretreatment unit, and adispenser capable of eluting the at least one substance or analyteretained by the pretreatment unit, wherein the hemolyzer is capable ofapplying shear forces sufficient to rupture cellular components of thewhole blood sample, and the apparatus is capable of operating thehemolyzer, pretreatment unit and dispenser such that hemolyzation,pretreatment and elution are performed in an automated fashion.